Hair analysis method

ABSTRACT

A method for the direct analysis of the presence of an analyte which becomes embedded in keratinized structures, e.g., hair, fingernails and toenails, from the bloodstream of a subject which comprises preparing a mixture containing dithiothreitol or dithioerythritol (“DTT”), an enzyme suitable for the digestion of the keratin structure and a sample of the keratin structure; permitting the enzyme to digest the sample of keratin structure to form a digest solution, followed by the addition of a salt of a metal of copper, zinc, manganese, iron, lead, cadmium, mercury, silver and cobalt to deactivate the DTT; and finally subjecting the digest solution to analysis to determine the presence of the analyte in the keratin structure sample. The protease enzymes papain, chymopapain, and proteinase K are preferred for use in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 08/813,376, filed Mar. 6,1997, now U.S. Pat. No. 6,022,693, which is a continuation-in-part ofU.S. application Ser. No. 07/737,703 filed on Jul. 30, 1991, nowco-pending which in turn is a continuation-in-part of U.S. applicationSer. No. 07/285,123 filed Dec. 16, 1988, now U.S. Pat. No. 5,324,642,which in turn is a continuation-in-part of U.S. application Ser. No.07/215,591 filed Jul. 6, 1988, now abandoned, which in turn is acontinuation-in-part of U.S. application Ser. No. 07/138,515, filedDecember 28, 1987, now abandoned.

FIELD OF THE INVENTION

This invention relates to an improved analytical method whicheffectuates the relatively rapid solubilization of hair and directanalysis of organic analytes, e.g., drugs of abuse, present in hair andother keratinized structures, e.g., fingernails and toenails, withouteffecting the structure of the analyte or being detrimental tobiological analyte probes, e.g., antibody, RNA/DNA and bio-receptorprobes. The analyte can be analyzed by adding the analyte probe directlyto the solubilized keratin structure containing the analyte to determinethe identity of the analyte as well as the extent and duration of itsconsumption by a subject.

BACKGROUND OF THE INVENTION

In the past, hair analysis techniques for the detection of trace metalswere developed that purported to provide information on an individual'snutritional status. One objection to the use of these techniques is thedifficulty of distinguishing between trace metals deposited in hair fromthe bloodstream and metals which have become embedded in hair throughexternal contact with, for example, water and cosmetic agents.Consequently, these techniques are not considered useful by the medicalcommunity for diagnosing nutritional problems, and therefore have notbeen considered sufficiently accurate to determine the level of aparticular trace metal consumed by a subject.

The problems with previous hair analysis techniques have caused relianceon urine and blood analysis techniques for the detection of ingestedchemicals, e.g., drugs-of-abuse, medications and toxic chemicals, in asubject. However, these techniques also are known to be disadvantageousin that the duration and intensity of use or exposure cannot beascertained. Urine and blood analysis, at best, can provide short terminformation concerning ingested drugs or chemicals such asdrugs-of-abuse. In addition, there are also problems with theinterpretation of such results. For example, the detection of a lowlevel of ingested chemical in the urine could mean that a subjectingested a small amount of the drug or chemical very recently or alarger amount several days earlier. Thus, chronic drug use cannot bedetermined with these methods without repeated testing.

In response to the problems of establishing a reliable and accuratemethod that would measure both the duration and intensity of use ofdrugs-of-abuse, medications, toxic chemicals, etc., work performed byDr. Werner A. Baumgartner, as reported in “Radioimmunoassay of Hair forDetermining Opiate Abuse Histories”, J. Nucl Med 20:749-752 (1979),determined that long-term histories of exposure to drugs-of-abuse can beobtained through the analysis of mammalian body hair, since thesesubstances are “trapped” within individual hair fibers during theirsynthesis. In this respect, hair was shown to act like a tape recorder,i.e., past exposure histories can be evaluated through sectionalanalysis of hair samples. It was found that heroin, once in thebloodstream, will find its way into hair as it is synthesized.

Thus, it was discovered in this study and confirmed by subsequentstudies that a variety of chemicals, such as drugs-of-abuse,medications, toxic chemicals, etc., hereinafter collectively referred toas “analyte”, are trapped by hair during its synthesis and that thesesubstances are “locked up” in hair for essentially the duration of thehair. This was found to be true for head and body hair as well as forother keratinized structures such as fingernails. Suzuki et al.,Forensic Sci. International, 24:9-16, 1984. These entrapped substancescannot be washed out of hair, and are completely released only upon thecomplete, or nearly complete, destruction of the hair fiber.

Prior art methods of extracting an analyte from hair included subjectingthe hair to hot methanol solutions (Baumgartner et al., J. Nucl Med 20,748, 1979) and by overnight incubation of hair in an alkaline or acidmedium. D. Valente, et al., Clinical Chemistry, 1952, Vol. 27, No. 11,1981. Prior methods also include the use of a mortar and pestle torelease the entrapped analyte in conjunction with a solvent.

However, solvent extraction procedures suffer from several problems inaccurately determining the presence and amount of an ingested analyte.One of these problems is that the solvent extraction methods frequentlyremove only a small unknown and variable fraction of the total analytepresent in the hair sample. Such methods also tend to be time consuming,and generally involve elevated temperatures which may damage theanalyte. Another disadvantage is that different analytes requiredifferent solvents for extraction. For example, a hair sample containingmorphine, phencyclidine (“PCP”), cocaine and marijuana has to beextracted sequentially with several different solvents, which is a verytime consuming procedure, particularly since the frequently toxicsolvents have to be evaporated in expensive fume hoods before analysiscan proceed.

Other methods and studies pertaining to the degradation of hair and hairanalysis include:

O. Suzuki, et. al., in a publication by Elsevier Scientific PublishersIreland Ltd., discloses a method for detecting methamphetamine andamphetamine in nail clippings or hair in which the substance was firstwashed in a mixture of methanol and water and dissolved in sodiumhydroxide, followed by analysis of the extracted drug.

A. W. Holmes, in Textile Research Journal, 706-712, August 1964,discloses the degradation of human hair by papain using sodium sulfiteas enzyme activator.

Annette M. Baumgartner, et al., in the Journal of Nuclear Medicine,20:748-752, 1979, discloses the extraction of morphine and heroin fromhair by pulverizing hair with a mortar and pestle followed by treatmentwith methanol.

D. Valente, et al., in Clinical Chemistry, Vol. 27, No. 11, 1981,discloses Dr. Baumgartner's technique of subjecting hair to a treatmentof hot methanol to effectuate extraction of drugs of abuse as well asthe author's technique of extracting morphine in an acid or alkalinemedium.

A. M. Baumgartner, et al., in Journal of Forensic Sciences, p. 576-81,July 1981, discloses the extraction of PCP with mortar and pestlefollowed by treatment with methanol. The extracted PCP was then analyzedwith RIA.

Smith et al., in Journal of Forensic Sciences, Vol. 26, No. 3, July1981, pp. 582-586, disclose the testing of hair for the presence ofphenobarbital, in which a single head hair was washed, dried, cut in 2mm lengths and added to 0.2 ml 0.1% SDS/saline solution, and a sampleassayed by radioimmunoassay.

W. A. Baumgartner, Black. et al., in J. Nucl Med 23: 790-892, 1982,discloses the extraction of cocaine from hair samples by refluxing thehair samples in ethanol followed by RIA analysis.

Ishiyama, et al., in Journal of Forensic Sciences, Vol. 28, No. 2, April1983, pp. 380-385, disclose a method whereby hair from methamphetamineaddicts was dissolved using 1.5 N hydrochloric acid at a pH between 1and 2, followed by analysis using a gas chromatograph and massspectrometry.

K. Puschel, et al., in Forensic Science International, 21 (1983)181-186, discloses the dissolving of hair samples by exposure to sodiumhydroxide and heat followed by analysis for the presence of morphine byRIA.

O. Suzuki, et al., in Journal of Forensic Sciences, Vol. 29, No. 2,April 1984, pp. 611-617, discloses the detection of methamphetamine andamphetamine in a single human hair by gas chromatography and chemicalionization mass spectrometry. The hair sample was first dissolved in asodium hydroxide solution to which was added N-methylbenzylamine.

N. J. Haley et al., in Clin. Chem. 31/10, 1598-1600 (1985), disclosesthe analysis of hair for nicotine and cotinine, in which washed hairsamples were dissolved in a buffer solution containing gelatin, sodiumchloride, Tris and EDTA, and adjusted to pH 7.4. Samples were thenanalyzed by radioimmunoassay.

Sramek, Baumgartner, et al., in A.M.J. Psychiatry 142:8, August 1985,discloses the analysis of hair samples of psychiatric patients withmethanol extraction and radioimmunoassay.

Baumgartner, et al., in Clinical Nuclear Medicine, Vol. 10, 4, September1985, discloses the benefits of extracting entrapped drugs of abuse fromhair followed by RIA analysis.

Gill, et al., in Nature, Vol. 318, p. 577 (1985) discloses the use of anSDS/proteinase k/dithiothreital mixture to extract DNA from whole blood,whole semen, vaginal fluid, hair roots, bloodstains and semen stains.The article states that “no DNA could be isolated from hair shafts”.

Smith et al., in J. Forensic Sci. 1986, 31(4), 1269-73, discloses thedetection of cocaine in perspiration, menstrual blood stains and hairusing RIA.

M. Margio, et al., in “Determination of Morphine and Other Opioids inthe Hair of Heroin Addicts by HPLC and MS/MS” at the InternationalConference, University of Verona, Jun. 25-26, 1986, discloses variousmethods to assay morphine from hair samples.

M. Marigo, et al., in the Journal of Analytical Toxicology, Vol. 10,July/August 1986, discloses a method for the quantitative determinationof morphine contained in the hair of heroin addicts, by means ofheat-acid hydrolysis, pre-column dansyl derivatization, straight phaseliquid chromatography and fluorescence detection.

Smith, et al., in Journal of Forensic Sciences, Vol. 31, No. 4, October1986, pp. 1269-1273, disclose a method for the analysis of hair for thepresence of drugs whereby hair samples were first washed, cut into smallsegments, mechanically pulverized for six minutes, refluxed in ethanoland the samples analyzed using radioimmunoassay.

M. Michalodinitrakis, Med.Sci.Law (1987), Vol. 27, No. 1, discloses thedetection of cocaine in rats from the analysis of hair samples, whichwere dissolved upon exposure to 1.5 N HCl, which brought the pH value to1-2, following incubation with 0.01 N Hcl at 37° C. for one hour.

Pelli, et al., in Biomedical and Environmental Mass Spectrometry, Vol.14, 63-68 (1987) discloses a procedure for the identification ofmorphine in the hair of heroin addicts in which hair is treated withdiethylether and hydrochloric acid followed by dissolution of the driedextract in methanol.

Higuchi et al., in Nature, Vol. 332, p. 543 (1988) disclose a method fordissolving hair at pH 8 by the action of dithiothreitol, proteinase K,and 2% sodium dodecylsulfate in order to extract DNA from the digest bya complex chemical extraction method.

Also noted are certain patents, e.g., U.S. Pat. Nos. 3,986,926,3,966,551, 3,939,040 and 3,623,950, which pertain to depilatory agentsfor the tanning of hides, and disclose the use of certain enzymes,including papain, in the dehairing process.

However, these and other prior art methods have proven disadvantageousfor the reasons noted above and/or because they degrade the analyteprobes (e.g., antibodies) of biological analytical methods, therebypreventing the use of such highly sensitive analytical techniques.

Thus, there exists a need for an analyte detection method that canrapidly and completely solubilize a certain analyte from keratinizedstructures of the body such as hair, fingernails and toenails of asubject and which permits direct analysis of the identity of the analyteand the duration of use of the analyte in, or exposure to, a subject,without destroying the analyte of interest and/or an analyte probe ofbiological analytical methods.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a drug and chemicaldetection method.

It is another object of the invention to provide a drug and chemicalhair analysis method.

It is another object of the invention to provide a reliable method ofdigesting head and body hair and other keratinized structures of thebody and directly analyzing the identity and amount of analyte containedtherein, and, where applicable, of determining the duration and extentof exposure of the analyte in a subject.

It is yet another object of the invention to provide a hair analysismethod that solubilizes an analyte from the inner core of hair withoutcausing damage to the analyte.

It is yet another object of the invention to provide a reliable hairdigestion and direct analyte detection method that effectively permitsthe use of highly accurate biological analytical methods such asradioimmunoassay.

It is yet another object of the invention to provide a reliable hairanalysis method that may be performed in a lesser period of time thanknown hair analysis methods.

It is yet another object of the invention to provide a drug detectionmethod effective for use in the drug testing industry standard five-drugscreen for marijuana, cocaine, opiates, methamphetamine andphencyclidine.

These and other objects are achieved by the novel analysis methodaccording to the invention, which comprises preparing a mixturecontaining dithiothreitol (DTT) or dithioerythritol (DTE), an enzymesuitable for the digestion of keratinized structures and a sample of akeratinized structure; permitting DTT or DTE to activate the keratinizedstructure and/or the enzyme; permitting the enzyme to at leastsubstantially digest the sample of keratinized structure to form akeratin digest solution; deactivating the DTT or DTE; and subjecting aportion of the keratin digest solution to analysis to detect theidentity and amount of the analyte, if present, in the keratinizedstructure sample.

The preferred keratinized structure is hair. The enzyme may be anyenzyme that, together with DTT or DTE, digests hair, and preferably is aprotease including papain, chymopapain, or proteinase K. In order toaccelerate the process, metal ions preferably in the form of metal saltsmay be added to the digest solution to deactivate any remaining DTT orDTE in the mixture which, if left active, would cleave the disulfidebond of the antibody of a biological analytical method such as animmunoassay, e.g. radioimmunoassay.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a method is provided thatpermits the rapid and complete digestion of head or body hair or otherkeratinized structure of an individual who may previously have ingestedone or more analytes, followed by the identification of the analyte byknown analytical biological probes such as the rapid and highlysensitive immunoassays. The release of the analyte into a digestsolution from the interior of hair is effectuated according to theinvention without damaging the analyte trapped within the organic matrixof the hair fiber which is to be analyzed, and without harmful effect ona subsequently-used probe (e.g., antibody) of a biological analyticalmethod. The invention also permits the detection of past use patterns ina subject over extended periods of time without performing repeatedtesting as is necessary in conventional testing methods which measurethe content of the analyte in samples of blood or urine. It has beenfound that the amount of analyte entrapped in hair of the sameindividual is directly proportional to the amount of analyte ingested.

A sample of a keratinized structure, e.g., hair, is first collected froma subject who may have ingested a particular analyte. Preferably, thehair sample is first washed by known methods to remove analyte or otherdrug or chemical which may have been deposited on the surface of thehair by external contact rather than by actual consumption. The hairsample is then subjected to treatment with a particular enzyme, togetherwith a particular enzyme/substrate activator, so as to effectuate thecomplete or nearly complete digestion of the organic matrix of the hairfiber, known as keratin. The subject analyte that has been “entrapped”within the organic matrix of the hair is then released into solution, oreven if protein bound, the analyte is accessible to the antibodyemployed in protein-based analytical methods. In order to fully andaccurately carry out the method according to the invention, a completedigestion of the sample is desirable.

Any enzyme that acts quickly to digest hair in conjunction with DTT orDTE without having a detrimental effect on the analyte is useful in theinvention. In this regard, proteases are preferred for use in theinvention. Most active, and therefore most preferred for use in theinvention, are the proteases papain, chymopapain and proteinase K.

A number of other proteases have been found to be effective in themethod according to the invention at low pH values (e.g., pH 7-9),namely, protease Type IV (bacterial, from Streptomyces caespitosus),Type VIII (from Bacillus subtilis), Type XI (proteinase K, fungal, fromTritirachium album), Type XIV (pronase, from Streptomyces griseus), TypeXVI (from Bacillus subtilis), Type XVIII (Newlase, from Rhizopusspecies), Type XIX (from Aspergillus sojae), Type XXI (from Streptomycesgriseus), Type XXIV (bacterial), Type XXVII (Nagarase), Type III(Prolase) and Type XXIII (from Aspergillus Oryzae) (all available fromSigma Chemical Co., St. Louis, Mo.).

As noted above, certain art-recognized procedures provide for the use ofpapain for use as a hair depilatory. These depilatory methods removehair from hides and skin by softening it sufficiently so as to permitits ready removal by scraping or other mechanical means, and utilizeinexpensive and less effective sulfhydryl enzyme and substrateactivators such as thioglycolic acid or cysteine. These methods onlypartly degrade the hair and do not provide for the complete chemicaldigestion of the hair. A mere softening of the hair does not lead to thecomplete, or nearly complete, digestion of hair which is necessary inorder to obtain a complete release of “entrapped” analyte. Moreover, thesulfhydryl enzyme activators used in these depilatory methods are alsoharmful to certain biological analyte probes such as antibodies.

In contrast to these depilatory methods, the method of the presentinvention utilizes “DTT” (2,3 dihydroxybutane-1,4-dithiol) or its isomer“DTE” (2,3 dihydroxybutane-1,4-dithiol) as the substrate and enzymeactivating agent. Surprisingly, it has been found that the use of DTT orDTE in the process of the invention significantly enhances the digestionof the sample within a relatively short period of time, e.g., aboutthree hours, resulting in the release of the analyte into the digestsolution. Particularly surprising is that the invention may be used inthe industry standard five-drug screen, in that it does not negativelyimpact upon any of those five drugs or their corresponding antibodies inthe analysis step of the invention.

This high activity of the enzyme is believed to be due, at least inpart, to the activation of the keratinized structure substrate itself byDTT and DTE, presumably by the action of DTT and DTE in opening updisulfide bonds in the keratinized structure, which facilitatesenzymatic attack.

Once the protein of the keratinized structure has been completely or atleast substantially digested, thereby releasing the analyte into thesolution mixture, it has been found to be necessary to deactivateDTT/DTE and the sulfhydryl enzymes prior to subjecting the analyte tobiological analytical probes, since the sulf-hydryl enzymes andenzyme/substrate activator(s) may interfere with the structuralintegrity of protein components of such methods.

The task of deactivating the sulfhydryl-dependent enzymes such as papainhas proven difficult since after the digestion step, the enzymes are“buried” in a “sea” of sulfhydryl groups belonging to the released hairproteins and enzyme/substrate activating agents. Known sulfhydrylblocking agents are ineffective in deactivating the enzymes, since theknown sulfhydryl blockers tend to bind to the degraded hair proteins andDTT or DTE and not necessarily to the enzyme sulfhydryl sites criticalfor blocking the activity of the enzymes. Thus, it is not possible toeffectively utilize the protein-based analytical methods if the enzymesulfhydryl sites are still active.

It was quite surprising, therefore, that DTT and DTE act not only toactivate enzymes and/or the keratinized structure substrate causingunexpectedly high hair digestion activity, but that they also may act todeactivate the enzyme by a direct or indirect (enzyme self-deactivation)mechanism after the enzyme effectuates the complete, or nearly complete,digestion of the hair protein. Typically, the enzyme deactivation occurswithin about four to five hours after exposure of the DTT or DTE to theenzyme, which is a sufficient amount of time for the enzyme toeffectuate the digestion of the hair sample. Once the enzyme has beendeactivated, it has been found that the enzyme cannot be reactivated orregenerated by exposure to fresh DTT or DTE.

Deactivation of at least certain of the non-sulfhydryl dependentproteases, e.g., proteinase K, by its inhibitor, phenylmethyl sulfonylchloride, is generally not required since the enzyme has not been foundto be active against the antibodies used in protein based immunoassaytechniques.

It also has been found that active DTT or DTE present in the hair digestsolution constitutes a hazard to the structure and activity of otherproteins to which it is exposed, e.g., antibodies utilized inradioimmunoassay. Thus, it was a further surprising result that DTT orDTE in the reaction mixture may not only act to deactivate the enzyme,but itself deactivates in the digest solution without the introductionof an inhibitor. Typically, DTT and DTE will deactivate after the hairsample has been digested, less than about 14 hours after its firstexposure to the enzyme depending on the various concentrations andamounts of the enzyme and DTT or DTE utilized, the pH, temperature,amount of hair sample, etc.

Thus, in accordance with the method of the invention, complete digestioncan be carried out in a relatively short period of time, e.g.,overnight, and the digest solution, which includes the released analyteof interest, can be directly subjected, effectively and accurately, toprotein-based ligand assay analysis methods the next morning. Typically,the entire method, from the washing of hair samples to theidentification of the analyte, should take no longer than about 16-20hours. Little or no intervention by the individual performing the methodis needed to release the analyte from the hair sample once the enzymeand DTT or DTE come into contact with the hair sample.

Alternatively, it has been discovered that the addition of certain metalions, typically in the form of metal salts, to the digest solutionresults in a rapid deactivation of DTT or DTE. The addition of lowamounts of such metal salts to the digest solution after digestion ofthe sample significantly accelerates the time in which the hair digestmixture can be subjected to the immunoassay method since it is notnecessary to wait for DTT or DTE to deactivate on its own. Thisdiscovery is particularly surprising as not all metal ions are effectivein deactivating DTT and DTE, or otherwise are not useful in theinvention.

Most effective for use in the invention are certain metal salts whichsurprisingly do not precipitate out of the solution after chemicallylinking with, and deactivating, DTT/DTE. It is important thatprecipitation not occur in the digest solution because suchprecipitation could result in a loss of analyte by adsorption.Preferably, precipitation is prevented by maintaining the pH of thedigest solution at about 6-8, and most preferably at about 7. One waythis may be accomplished is by the addition of one molar Trizma base.Surprisingly, the most preferable pH of about 7 is also the optimum pHfor the performance of radioimmunoassay (“RIA”).

In addition to Cu⁺⁺ salts (e.g., copper sulfate) as described inApplicant's U.S. Pat. Nos. 5,466,579 and 5,324,642, salts of Zn⁺⁺ (e.g.,zinc sulfate and zinc nitrate); Mn⁺⁺ (e.g., manganese sulfate); Fe⁺⁺(e.g., ferric sulfate and ferric chloride); and Fe⁺⁺⁺ (e.g., ferroussulfate) are particularly effective and preferred for use in theinvention. Also effective for use in the invention are salts of Pb⁺⁺(e.g., lead acetate and lead nitrate); Cd⁺⁺ (e.g., cadmium chloride);Hg⁺⁺ (e.g., mercuric chloride); Ag⁺⁺ (e.g., silver nitrate); and Co⁺⁺(e.g., cobalt chloride).

Typically, about 100 microliters of metal salt (10 mg/ml) is added to 1ml of hair digest solution about 4 to 5 hours after contacting theenzyme and DTT (or DTE) with the hair sample so as to permit the enzymeand DTT (or DTE) sufficient time to digest the hair sample.

Similarly, any salt of arsenite, and preferably sodium arsenite(NaAs0₂), may be utilized in the invention to remove residual DTT or DTEby formation of a precipitable compound. Typically, 100 microliters of a100 mg/ml solution of sodium arsenite is added to 1 ml of hair digestsolution to effectuate the deactivation of DTT and DTE. However,arsenite is not preferred because a precipitate usually develops. Itmay, however, be useful in certain circumstances.

Once the rapid and effective digestion of the sample occurs, the digestsolution may then be subjected to direct analysis by art recognizedprotein-based analytical methods such as RIA. Such methods are preferredfor use in the invention because RIA and related immuno- or ligandassays are currently the only known mass production procedures havingthe required sensitivity and convenience for measuring the lowconcentrations of analytes contained in hair samples. The use of thesemethods is preferred because only about 0.5 to 1.0 mg. of hair isnecessary for analysis by RIA and other protein-based analyticalmethods.

Other analytical methods may be utilized in place of or in addition tothe protein-based analytical methods, including instrumental means suchas chromatography, mass spectrometry, etc. In particular, these methodsmay be used to confirm positive results obtained in RIA. Because thesemethods are not protein-based, the steps of deactivation of the enzymeand DTT or DTE is not necessary when using non-protein-based analyticaltechniques. However, the speed and gentleness of the extraction methodaccording to the invention and the ability to quantitate the extractionefficiency through the inclusion of a “spike”, i.e., the inclusion of aknown amount of deuterated analyte, makes the presently discloseddigestion method also the method of choice for instrumental analysismethods such as gas chromatography and mass spectrometry.

The method according to the invention has been found to be effective indetecting the use and prior use of drugs of abuse such as cocaine,morphine/heroin, marijuana, phencyclidine or “PCP”, methaqualone andmethamphetamine. Moreover, the method according to the invention hasbeen found to be effective in determining prior usage of prescriptiondrugs such as digoxin, methadone and benzodiazepines. It is contemplatedthat any organic analyte present in the bloodstream of an individualwhich is transferred to the hair during its synthesis can be extractedand analyzed in accordance with the method of the invention.

In carrying out the method, it is preferred that an aqueous solution ofabout 110 mg DTT or DTE/10 ml water be used, although concentrations ofDTT or DTE of about 50-200 mg/10 ml water are effective in the method.It is preferred that the weight ratio of DTT or DTE to papain orchymopapain be about 110:2 [when enzyme purity is 16-40 BAEE units/mgprotein], although efficacious results have been observed at weightratios of DTT or DTE to papain or chymopapain ranging between about110:1 to about 110:4. With respect to proteinase K and other proteases,it is preferred that the weight ratio of DTT or DTE to proteinase K (orother proteases) be about 1200:1 (when enzyme purity is 10-20 units permg. protein), although weight ratios of 1200:0.5 to about 1200:2 alsowill be effective.

The concentration of hair protein is preferably kept constant at about10 mg hair/cc of digest solution so as to prevent variable matrixeffects in a subsequently utilized protein-based analytical method.

The enzymatic digestion of hair and other keratinized structures,according to the method of the invention, may be conducted at lowtemperatures and near neutral pH. When papain, chymopapain or othersulfhydryl dependent enzyme is utilized as the enzyme, the method may beperformed at a temperature of between about 20° C. and 40° C., and at apH between about pH 8.8 and 10.5. Preferably, the pH of the method isbetween about 8.8 and 9.5 at a temperature of about 37° C.

When proteinase K or other proteases are utilized as the enzyme, it ispreferable to perform the method between about 20 and 40 degreescentigrade and at a pH between about 7 and 9. When the temperature isabout 37 degrees centigrade and the pH about 7.0 or below, the risk ofaltering the structure of a particular analyte is at a minimum. Otherenzymes which digest hair under neutral or acid conditions include:Protease Type XIV (Pronase), Type III (Prolase), Type IV, Type VIII,Type XVI, Type XVIII, Type XIX, Type XXIV, Type XXVII (Nagarse), TypeXXVIII, Type XXI and Type XXIII.

Under certain circumstances, it is advantageous to perform the methodaccording to the invention at a lower than usual pH in order to preservethe chemical structure of the analyte. As stated above, the digestiontypically will occur at a pH between about 8.8 and 10.5 when asulfhydryl dependent enzyme (e.g., papain) is utilized and between 7 and9 when a protease such as Proteinase K is utilized. At any pH in eitherof these ranges, however, certain analytes may become unstable orhydrolyze to a different form, which may impact on the measure of bothquantity and quality of the analyte in the subsequent analysis step.

Thus, for example, at a pH of about 7, the heroin metabolite,6-monoacetylmorphine, breaks down rapidly to morphine, thereby makingheroin users indistinguishable from morphine users. In addition, thestability of cocaine is also quite pH dependent, and the performance ofthe method at an improper pH may lead to a false interpretation of apositive cocaine result.

Ingested cocaine naturally hydrolyzes to benzoylecgonine in the bloodand eventually ends up entrapped in hair both as cocaine andbenzoylecgonine. The digestion of hair from a cocaine user will lead tothe release of both cocaine and benzoylecgonine from the hair intosolution thereby resulting in the conclusion that a positive cocainehair analysis result was caused by cocaine ingestion.

In situations where an individual has not ingested cocaine but is onlyexposed to cocaine environmentally, contaminated hair will contain onlycocaine and not the benzoylecgonine metabolite. Thus, the absence ofbenzoylecgonine confirms lack of drug use. Conversely, the presence ofbenzoylecgonine refutes any claim that a positive result was caused bythe external contamination of a hair sample, i.e., by faulty,ineffective washing of the sample by the laboratory to remove cocainecontaminants deposited from the environment. However, cocaine tends tohydrolyze to benzoylecgonine at a pH above about 6.5 and a temperatureof 37° C. As a result, the certainty of distinguishing between drug useand external contamination can only be achieved if the pH of the digestsolution is maintained so as to avoid the production of significantquantities of benzoylecgonine.

Thus, in the case of certain analytes such as cocaine which may bechemically altered by a higher pH, it is desirable to perform the methodof the invention at a pH which avoids hydrolysis or other chemicalreaction of externally deposited analyte which inadvertently has endedup in the digest solution. In the case of cocaine, performance of themethod at a pH below about 6.6 at 37° C. will ensure that thebenzoylecgonine in the sample is directly related to ingested cocaineand not to externally deposited cocaine.

According to the invention it has been found that certain biologicaldetergent compounds useful for solubilizing biological membranecomponents aid in the digestion of hair at a relatively low pH while notinterfering with enzymatic activity or the antibody-antigen reactionwhich will influence the sensitivity of the immunoassay. This issurprising and unexpected since other biological detergents have beenfound to be unsuitable for use in the invention because they areineffective in aiding digestion at the desired low pH, they deactivateproteinase K or other hair protein digestion enzymes and/or they impacton the binding of the analyte by the antibody thereby drasticallyreducing the sensitivity of the immunoassay. See, e.g., Higuchi, R. etal., “DNA Typing From Single Hairs”, Nature, 332:543-546, 1988.

These biological detergents, together with an appropriate enzyme (e.g.,protease and sulfhydryl enzymes) and activator (e.g., DTT and DTE), areeffective in aiding the digestion of the hair sample at a lowered pH inthe range of about 5.8 and 8. Those detergents found to be useful in theinvention include the bile acid detergents, such as glycocholic acid,cholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid,taurodeoxycholic acid and salts thereof, including sodium salts. Otherdetergents effective for use in the invention are sulfo-betaines, suchas the Zwittergents®, and betaines, such as Empigen BB (N-dodecyl-N,Ndimethylglycine) (all available from Calbiochem Corp., La Jolla,Calif.).

Still other detergents which are useful in aiding the digestion of hairaccording to the invention at a relatively lower pH are thealkylglucosides, including hexyl-β-D-glucopyranoside,heptyl-β-D-glucopyranoside, octyl-β-D-glucopyranoside,nonyl-β-D-glucopyranoside, decyl-β-D-glucopyranoside,dodecyl-β-D-maltoside and octyl-β-D-thioglucopyranoside (OSGP). Mixturesof alkylglucosides, such as the product ELUGENT (Calbiochem), are alsoeffective.

Particularly preferred for use in the invention are the bile acidscholic acid and glycocholic acid which aid in the digestion of hair at apH in the range of about 6.3-8. The deoxycholates such as deoxycholicacid and glycodeoxycholic acid are effective in aiding in the digestionof hair at a pH above about 7.

As discussed above, the hydrolysis of cocaine occurs at a pH of about6.6 and above at 37° C. Thus, to avoid significant hydrolysis of cocaineto benzoylecgonine, the digestion preferably is performed at a pH of 6.5or below, and typically in the range of about 6.3-6.5.

Surprisingly, certain of these detergents are efficacious when theindustry standard five-drug screen for the most common drugs of abuse inthe United States, i.e., marijuana, cocaine, phencyclidine,methamphetamine and opiates, is performed using the method of theinvention. Thus, they do not impact on any of the analytes or antibodiesinvolved in the five-drug screen, and do not result in false negativesor positives. This is particularly surprising given the fact that thechemical nature of these five analytes ranges from highly lipidic drugssuch as PCP and marijuana to highly water soluble drugs such asbenzoylecgonine and morphine.

The particular detergents most effective for use in the five-drug screenare cholate, deoxycholate, cholic acid, deoxycholic acid,octyl-β-D-glucopyranoside and octyl-β-D-thioglucopyranoside. The bileacid detergents, alkylglucosides, sulfobetaines and betaines are mostpreferred when a screen is performed that includes cocaine, opiates,phencyclidine and methamphetamine. In a screen solely for cocaine, thepreferred detergents are cholic acid, Zwittergents®, alkylglucoides, andN-dodecyl-N,N dimethylglycine.

Of the sulfo-betaine detergents manufactured by Calbiochem Corp. of LaJolla, Calif., Zwittergent® SB3-14 (CAS Registry No. 14933-09-6,N-tetradecylsulfobetaine or 3-(dodecyldimethylammonio)propane-1-sulfonate.) is preferred. Digestion of hair using theZwittergent® sulfo-betaine detergents typically occurs at about pH 6.3at 37° C. Zwittergents® are of the class of detergents known assulfo-betaines having the general structure:

wherein x may be any number which provides an effective biologicaldetergent. Preferred are those compounds wherein x is in the range of7-16. Most preferred is the detergent when x is 14. Other Zwittergent®sulfo-betaine detergents useful in the invention include:

1. Zwittergent® SB3-08 wherein x=7 [N-octyl sulfo betaine or3-(Octyldimethylammonio) propane -1-sulfonate] [CAS # 15178-76-43].

2. Zwittergent® SB3-10 wherein x=9 [N-D-dodecylsulfobetaine or3-(dodecyldimethylammonio) propane-1-sulfonate] [CAS Registry #15163-36-7].

3. Zwittergent® SB3-12 wherein x=11 [N-dodecylsulfobetaine or3-(Dodecyldimethylammonio) propane-1- sulfonate] [CAS #14933-09-6].

4. Zwittergent® SB3-14 wherein x=13 [N-tetradecylsulfobetaine or3-(Dodecyldimethylammonio) propane-1-sulfonate] [CAS # 14933-09-6].

5. Zwittergent® SB3-16 wherein x=15 [N-Hexadecylsulfobetaine or3-(Hexadecyldimethylammonio) propane-1-sulfonate] [CAS #2281-11-0]

The bile acid detergents and Calbiochem Zwittergents® SB3-8, SB-10,SB3-12, SB3-14 and SB3-16 (x in the range of 7-16) are preferred ineffectuating the digestion of hair at a relatively lower pH which isdesirable in methamphetamine, PCP and opiates assays. The cholate andCalbiochem Zwittergents® are preferred for use in the cocaine assay. Thecholate and deoxycholate detergents are preferred for use in marijuanascreening assays.

In practice, the biological detergent is mixed with the aqueous digestsolution of the activator such as DTT (or DTE) and the enzyme(preferably proteinase K) prior to contact of the solution with the hairsample at a preferred temperature range of about 30-40° C. as describedherein. Typically, about 1-2 mg of biological detergent is added toabout 1 cc of digest solution.

In another embodiment according to the invention, an ion exchange resinis employed to remove from the hair digest solution a substance whichuniquely interferes with the marijuana assay. This interference hasoccurred with all currently available commercial RIA kits suitable fordetecting cannabinoids. The interfering substance, effectively presentin every hair sample in varying amounts from individual to individualand believed to be naturally occurring in hair, appears to interferewith the assay as a result of a cross reaction with the marijuana RIAantibody (i.e., specific to cannabinoids) rather than by matrix effects.This appears to be the case because dilution of the interferingsubstance produces an asymptotic curve which appears identical in shapeto the calibration curve obtained. with the carboxytetrahydro-cannabinol(“carboxy-THC”) standard used for an RIA marijuana assay, rather thanproducing an S-shaped dilution curve which would have been expected ifmatrix effects were the cause of the interference. It appears that thisinterfering substance is lipidic and bears a close resemblance instructure at the immunological binding site to carboxy-THC.

Because of its similarity to carboxy-THC, and other cannabinoids such astetrahydrocannabinol (“THC”), the interfering substance results in falsepositive results in assays on hair digests using RIA to determinemarijuana exposure. In other words, the RIA erroneously will identifythe interfering substance as a cannabinoid from exposure to marijuana,even in individuals not exposed to marijuana. Thus, it is necessary whenperforming an RIA assay for marijuana exposure to somehow remove theinterfering substance from the digest solution prior to subjecting thesolution to immunoassay analysis.

Since the presence of this interfering substance in hair is a newdiscovery, there is no method known to the art for removing it. Removalof the interfering substance from the digest solution is furthercomplicated by its similarity to lipidic carboxy-THC and THC, the morecommon diagnostic analytes in a marijuana assay. Many filteringtechniques with the capability of filtering out undesirable substancesare not effective in removing the interfering substance from the digestsolution, because they either do not effectively remove the interferingsubstance, and/or because they remove the analytes, e.g., THC andCarboxy-THC.

It was thus surprising that there exists in hair an analyte indicativeof marijuana exposure which does not possess many of the same lipidicproperties of the interfering cross reacting substance but yet willreact with the cannabinoid antibody used in the RIA assay for marijuana.The exact chemical structure of this immunoreactive substance(s), ormodified-lipidic marijuana analyte, is unknown. However, its presence inthe hair sample will indicate marijuana use.

Also surprising is the discovery that the interfering substance may beremoved from the digest solution without removing the newly discoveredmodified-lipidic marijuana analyte by the use of certain commerciallyavailable ion exchange resins. It has been found that suspensions ofcertain ion exchange resins upon contact with the digest solution willremove the interfering substance along with certain cannabinoids such asTHC from the digest solution, but will leave in the digest solutionother diagnostic cannabinoids, such as the modified lipidic marijuanaanalyte, which then may be detected by commercially available RIA kitsutilizing a cannabinoid antibody. The use of these ion exchange resinsto remove the interfering substance, thus permitting the detection ofthe modified-lipidic marijuana analytes in the RIA assay withoutinterference, is both convenient and cost effective.

Effective ion exchange resins are both anionic and cationic. Theygenerally are commercially available, such as from Sigma Chemical Co. ofSt. Louis, Mo. However, they have been found to be most effective not inthe form in which they are commercially available (generally course,fast settling particles), or in the way they are generally used (e.g.,packed columns) but when contacted with the digest solution in the formof a suspension, e.g., when broken apart into much smaller, slowlysettling particles by vigorous stirring and made into a fine suspensionof these small particles. Effective ion exchange resins include:

1. Anion exchangers on dextrose such as DEAE Sephadex® (A-25 and A-50Diethylaminoethyl Sephadex®) and QAE Sephadex® (Q-50Diethyl-[2-hydroxypropyl] aminoethyl Sephadex®);

2. Anion exchangers on agarose such as DEAE Sepharose® CL-6B(Diethylaminoethyl Sepharose®) and Q Sepharose®;

3. Anion exchangers on cellulose such as DEAE-Sephacel®(Diethylaminoethyl Sephacel®); Ecteola Cellulose (EpichlorohydrinTriethanolamine Cellulose); PEI Cellulose (Polyethyleneimine Cellulose);QAE Cellulose (Diethyl-[2-Hydroxypropyl]aminoethyl Cellulose); and DEAEcellulose;

4. Cation exchangers on dextran, such as SP Sephadex® C25 (SulfopropylSephadex®)

5. Strongly acidic cation exchangers on polystyrene, such as Amberlite®200 (active group: sulfonic acid, sodium form); Dowex® HCR-S (activegroup: nuclear sulfonic acid, hydrogen form) and Dowex® macroporousresin (active group: nuclear sulfonic acid, hydrogen form).

6. Specialty Exchangers such as benzyl DEAE cellulose (BenzylDiethylaminoethyl cellulose) and TEAE cellulose(triethylaminocellulose).

The concentration of the resin will vary depending on the resinemployed. Concentrations may vary up to about 50% (wt./vol.). Forexample,.a suspension of DEAE Sephadex® A25 in deionized waterpreferably is prepared using about 3 to 9 gm. resin/100 ml water. Thesuspension is allowed to swell. At least one hour of swelling at roomtemperature has been found to be adequate. After swelling, thesuspension is stirred or shaken vigorously for at least thirty minutesand preferably up to about sixty minutes until a very fine suspension isobtained.

Preferably, the suspension has a settling time in the range of 50-60minutes compared to about 10-15 minutes of an aqueous suspension of anunshaken resin. Vigorous stirring or any other method which results inthe break up of the resin into small particles provides the optimumresults in removing the interfering substance from the digest solution.

To achieve removal of the interfering substance, approximately equalparts of hair digest solution and fine suspension are mixed together.The ratio of suspension to hair digest solution, however, will varydepending on the resin used and its concentration in the resinsuspension. Generally, the ratio of digest solution to suspension is 4to 3, if a suspension of 3 to 9% (wt./vol.) is used. The mixture isrotated so that the suspension and digest solution are in directcontact. Distilled water is then added, the mixture centrifuged and asample of the supernatant from the resinated digest solution assayedaccording to the invention.

While the method according to the invention for removing the interferingsubstance has been described in connection with a digest solution, it isrecognized that the ion exchange resin suspension may be used in anyhair analysis method where removal of the interfering substance isdesirable or necessary, provided that the analytes are ultimatelycontained in an aqueous albumin solution near neutral pH as required forthe RIA assay. The resin suspensions may be used, for example, togetherwith known hair extraction (acid, base or solvent) methods or any othermethod which disrupts a hair sample and results in the release into asolution at least a portion of the contents of a hair sample whichincludes the interfering substance.

In contrast to other available analyte detection methods such as urineand blood analysis, the method in accordance with the invention permitsdetection of exposure to an analyte over a period of time, and istherefore quite beneficial in detecting chronic drug use. Since hair isknown to grow at a rate of about 0.3-0.4 mm/day or about 1.0-1.3cm/month, it is possible to measure consumption or exposure as far backas the hair length permits by evaluating snippets of hair of variouslengths, and the use of highly sensitive protein-based analyticalmethods permits analysis of small samples of analyte contained in thesmall snippets of hair.

Through sectional analysis, the method of the invention provides arelatively permanent record and evidence of a pattern of drug use, orthe prior ingestion of other substances, for periods ranging fromseveral days to months or even years after last use. The history of suchexposure can be made as detailed as desired by analyzing suitably shortsections of hair representing different periods of growth. In this way,prior usage over time, and the extent of such use, can be determined.

Although the use of head hair is preferred for use in the invention dueto its length and accessibility, it is possible to utilize any otherbody hair or fingernails in the method of the invention. Thus, it is notpractically possible to evade testing by the method of the invention byshaving one's head or body hair.

However, treatments such as perming and dyeing may increase the rate ofdigestion of hair subjected to the method according to the invention. Insome cases, some analyte may be lost prior to performing the proceduredue to such treatments. When the subject hair has been so altered, anincrease in digestion rate is evident and an appropriate correctionfactor may be applied based upon known rates of normal hair digestion.

Certain other cosmetic agents, such as certain relaxing agents, maycause hair to become resistant to digestion. Such resistance may beovercome in some instances by increasing the quantity of enzyme to beused. Preferably, proteinase K is utilized as the enzyme when suchresistance to digestion is encountered.

Alternatively, when it is not possible to make use of body hair or insome instance when the use of hair is not desirable, the use of otherkeratinized tissue such as fingernails and toenails may be used in theinvention. In this regard, the effective ratio of DTT or DTE to enzymeneeded to digest fingernails and toenails in order to release theanalyte is about the same as for use with hair. Once the fingernail ortoenail samples are digested in accordance with the method describedherein, the released analyte may be analyzed by a desired analyticalmethod.

In another aspect of the invention, it has been surprisingly discoveredthat melanin granules contained in hair can be dissolved by the combinedaction of the enzyme (preferably papain), DTT or DTE and ethylenediamine tetraacetic acid (EDTA), the latter at a concentration of about5 mg EDTA/ml of digest solution. Since certain analytes or drugs ofabuse such as PCP have been discovered to accumulate in the granulescontained in mouse hair, dissolution of the granules, which also arepresent in the digest solution of human hair, may possibly beeffectuated and the analyte contained in the human hair granuleidentified.

In accordance with this aspect of the invention, a hair digest solutionis obtained as described above, and the melanin granules recovered fromthe hair digest solution, e.g., by centrifugation. The melanin granulesare then contacted with EDTA, the enzyme and DTT or DTE to release theanalyte from the melanin granules, and the analyte analyzed by themethods described above.

The benefits to be obtained from use of the method according to theinvention are many. The method provides a prompt and accurate diagnosisof prior exposure to a particular analyte. The subject hair andkeratinized structure analysis method can provide a record ofconsumption, or non-consumption, over very long periods of time. Guesswork regarding the true significance of one blood or urine analysis willbe eliminated. Hair collection is less intrusive and less physicallyrepulsive than blood or urine collection, and samples cannot be alteredor substituted, nor can detection be evaded by short term abstention or“flushing” (excessive fluid intake) prior to a scheduled testing, e.g.,pre-employment test or annual physical examination. Samples may bestored indefinitely without refrigeration.

The methods according to the invention, useful for the digestion ofkeratinized structures, e.g., hair, can also be used to ascertain thepresence and structure of naturally occurring components of hair such asDNA.

The following examples illustrate certain aspects of the invention butthey do not limit the invention as set forth in the specification andclaims.

EXAMPLE 1

Extraction of Cocaine from Hair Sample

10 mg of hair was removed from a subject suspected of being a cocaineaddict and washed by shaking in water at 37° C. for 30 minutes. To 10ml. of distilled water, 110 mg. of dithiothreitol(2,3-dihydroxybutane-1,4-dithiol, Cleland's reagent, obtained from SigmaChemical Co., St. Louis, Mo.), was added. The pH of the solution wasadjusted to pH 9.1 with 15% potassium hydroxide added dropwise withstirring of the DTT solution. Stirring was continued while adding 80microliters of Type III papain solution (papainase EC 3.4.22.2)(obtained from Sigma Chemical Co., 16-40 BAEE units activity per mg.protein). The enzyme solution was at a concentration of 30 mg of enzymeprotein/ml of water, where 1 mg of enzyme protein has an activity of16-40 BAEE units [one BAEE unit will hydrolyze 1.0 micromole of sodiumbenzoyl-L-arginine ethylester at pH 6.2 at 25° C.].

To 1 ml of this solution was added the 10 mg hair sample in a 13×75 mmpolycarbonate test tube. The solution was incubated in a 37° C. waterbath with shaking for 2 hours, and the solution was allowed to standovernight at 37° C. without shaking. The solution containing thedissolved hair sample was centrifuged at 2,000 rpm [Damon IEC model CRU5,000 centrifuge] to remove the melanin granules. To 1 cc of the hairdigest solution was added 200 microliters of a 1 molar phosphate buffer,pH 5.5.

100 microliters of this solution was assayed by RIA for the presence ofcocaine [benzoylecgonine equivalent, or “BEE”]. RIA analysis revealed83.6 nanograms BEE/10 mg of hair.

EXAMPLE 2

Addition of Dithioerythritol

The hair sample of Example 1 was analyzed using the digestion and assayprocedure set forth in Example 1, except for the replacement ofdithiothreitol (DTT) by dithioerythritol (DTE). The sample was assayedby RIA, which revealed 82 nanograms cocaine (BEE) per 10 mg of hair.

EXAMPLE 3

Addition of Cupric Sulfate

After digesting the hair sample in the water bath for four hours, 100microliters of a 10 mg/ml cupric sulfate solution was added to 1 ml ofthe hair digest solution prepared as set forth in Example 1. Thesolution was shaken at 37° C. for about 30 minutes prior to the additionof phosphate buffer and assay by RIA. One hundred microliters of thehair digest solution was subjected to RIA analysis, which revealed 85.0nanograms of cocaine (BEE)/10 mg of hair.

EXAMPLE 4

Addition of Metal Salts to Deactivate DTT

A solution was prepared containing 100 ml water, 2.09 g BIS-Tris (SigmaChemicals), 0.2 g cholic acid, 1.2 g dithiothreitol, and 200 unitsProteinase K. The pH was adjusted to 6.5. One milliliter of the solutionwas then added to each 10 mg of a hair sample. After centrifugation ofthe digested hair, to 1.0 ml of the supernatant was added 50 μl of 1.0 MTrizma base and then 100 μl of a 1% metal salt solution (0.04 M exceptwhere indicated below). The digests were then assayed in themethamphetamine RIA.

The methamphetamine assay was performed as follows: 100 μl of the digestwas placed in 12×75 mm test tube, followed by 300 μl of¹²⁵I-methamphetamine tracer and 100 μl of anti-methamphetamine (sheep)antibody. The mixture was incubated at room temperature for 1 hour atwhich time 200 μl of a second antibody (donkey anti-sheep) was added andthe mixture incubated for another half hour. The tubes were thencentrifuged for 20 minutes at about 3500 RPM. After decanting, theremaining precipitates were counted in a gamma counter.

The results in the tables below show the B₀'s of the digests withvarious metals against a control with no metal expressed as percent ofthe B₀ observed with copper sulfate, or a metal already shown to beequivalent to copper as a DTT/DTE neutralizing agent.

B_(o) as % of B_(o) with Copper Sulfate METAL in Methamphetamine AssayEXPERIMENT 1. Copper Sulfate (CuSO₄.H₂O, .08 M) 100 Zinc Chloride(ZnCl₂, .08 M) 111 Cadmium Chloride (3CdSO₄.8H₂O, .08 M) 97 No Metal 14EXPERIMENT 2. Copper Sulfate (CuSO₄.5H₂O) 100 Ferric Sulfate (Fe₂SO₄) 98No Metal 30 EXPERIMENT 3. Copper Sulfate (CuSO₄.5H₂O) 100 ManganousSulfate (MnSO₄) 103 Zinc Chloride (ZnCl₂) 100 No Metal 30 EXPERIMENT 4.Copper Sulfate (CuSO₄.5H₂O) 100 Mercuric Chloride (HgCl₂) 80 SilverNitrate (AgNO₃) 86 Cobalt Chloride (CoCl₂.6H₂O) 68 No Metal 30 B_(o) as% of B_(o) with Cadmium Chloride METAL in Methamphetamine AssayEXPERIMENT 5. Cadmium Chloride (3CdSO₄.8H₂O) 100 Lead Nitrate (Pb(NO₃)₂)107 Lead Acetate (Pb(Ac)₂.3H₂O) 110 No Metal 45 B_(o) as % of B_(o) withFerric Sulfate METAL in Methamphetamine Assay EXPERIMENT 6. FerricSulfate (Fe₂SO₄) 100 Manganous Sulfate (MnSO4) 100 Ferrous Chloride(FeCl₂.4H₂O) 95 Ferric Chloride (FeCl₃) 98 Ferric Chloride (FeCl₃.6H₂O)96 Ferric Sulfate (Fe₂SO₄.7H₂O) 99 No Metal 29

EXAMPLE 5

Addition of Sodium Arsenite

After digesting the hair sample in the water bath for four hours, 100microliters of a 100 mg/ml sodium arsenite solution was added to 1.0 mlof the hair digest solution prepared as set forth in Example 1. Thesolution was shaken at 37° C. for 30 minutes. 200 microliters of 1M, pH6.5, phosphate buffer was added prior to assay by RIA. A precipitate wasobserved. One hundred microliters of the hair digest solution wassubjected to RIA analysis, which revealed 82 nanograms of cocaine (BEE)per 10 mg of hair.

EXAMPLE 6

Substrate Activation by Dithiothreitol (DTT)

10 mg of hair were exposed to 11 mg of DTT at pH 9.1 for a period of 20hours. The DTT solution was removed and replaced with DTT and papain asin EXAMPLE 1. The hair specimen dissolved within 10 minutes as comparedto within one hour for a control specimen not pretreated with DTT anddigested as in EXAMPLE 1, thereby demonstrating that DTT activated notonly the sulfhydryl-dependent enzyme, papain, but the enzyme substrate,hair, as well.

EXAMPLE 7

Digestion of Fingernails

A 10 mg. sample of fingernail clippings was obtained from a subject, andsubjected to a detergent wash. 220 mg of DTT was added to 10 ml of waterin a test tube and the pH adjusted to pH 9.1 as in Example 1. A papainsuspension, 160 microliters, was then added. 1.0 ml of this solution wasthen added in a test tube to 10 mg of fingernail clippings and shaken at37° C. for a period of 24 hours until dissolution occurred. The digestsolution was then analyzed by RIA as previously described.

EXAMPLE 8

Performance of Sectional Analysis

A hair sample, about 6 cm in length, was obtained from an individualsuspected of being a heroin addict. The samples were carefully sectionedinto three 2 cm sections, with corresponding sections added to threeseparate test tubes and washed. The hair samples were subjected to theprocess described in Example 1, except that chymopapain (EC 3.4.22.6)was used in place of papain as the enzyme. The samples were agitatedovernight as previously described.

RIA analysis revealed morphine content in the three sections of 13.5,5.7 and 0 nanograms/10 mg hair.

EXAMPLE 9

Digestion of Digestion-Resistant Hair

Ten milligrams of hair which had been treated with relaxer was incubatedovernight in the solution digest described in Example 6. The hair sampledid not dissolve in the usual 20-hour period. A greater and additionalamount of proteinase K, i.e., 1 mg, was then added to the partiallydigested sample. The sample then dissolved within the next 24 hours. Thedigest was centrifuged and 100 μl CuSO₄ solution (10 mg/ml) was added to1 ml of the supernatant which was then shaken at 37 degrees centigradefor 30 minutes. 200 μl of 1 M phosphate buffer pH 7 was added. Due tothe high amount of proteinase K in the resulting digest, 20 μl of theinhibitor phenylmethyl sulfonyl chloride in ethanol was added to thedigest prior to assay by RIA.

RIA analysis revealed 7.4 ng cocaine/10 mg hair.

EXAMPLE 10

Digestion and Analysis of Hair Using Proteinase K and Cholic AcidDetergent

Separate samples of 10 mg of normal hair and 10 mg of hair containingthe cocaine metabolite benzoylecgonine (BEE) were collected and placedin separate 12×75 mm polycarbonate tubes. One ml of the followingsolution was added to the hair in each of the tubes: 0.5M Tris buffer(pH 6.5 at room temperature) containing one ml 2 U Proteinase K, 20 mgcholic acid (sodium salt) and 60 mg dithiothreitol. The mixtures wereshaken 16 hours at 37° C. After 16 hours, the samples were centrifugedat 3,000 RPM for 20 minutes. To 0.9 ml of the supernatant removed aftercentrifugation was added 10 μl of phenylmethylsulfonyl fluoride (6% inEthanol). This was mixed, followed by the addition of 90 μl CuSO₄.5H₂O(10 gm/liter) to each solution. This mixture was shaken at 37° C. for 30minutes. 100 μl of this mixture was then assayed by RIA. Equivalentsolutions as described but containing no cholic acid detergent and nohair protein were also assayed. The results were as follows:

A. Sample of hair protein not containing cholic acid or BE: 14,686 cpm(counts per minutes on gamma counter) (B_(O)).

100 μl sample of hair protein containing 0.14 ng BE and no cholic acid:11,128 cpm (B), or a

B/B_(O) of 76% (indicating the presence of BE).

B. 100 μl sample of hair containing cholic acid but no BE: 12,048 cpm(B_(O))

100 μl sample of hair containing 0.14 ng BE and cholic acid: 8,602 cpm(B), or a

B/B_(O) of 71% (indicating presence of BE)

The similarity of the resulting B/B_(O)'s (76% and 71%) with and withoutcholic acid indicate that cholic acid did not interfere with the assayfor cocaine or benzoylecgonine.

EXAMPLE 11

Digestion of Hair Using Proteinase K and Removal of InterferingSubstance in Marijuana Assay

10 mg of hair from a known normal subject (not marijuana users) and 10mg of hair from 1 known marijuana user were placed in separate 13×75 mmpolycarbonate tubes. One ml of the following solution was added to thehair in each of the tubes: 0.5M Tris buffer (pH 6.5 at room temperature)containing in one ml 2 U Proteinase K, 2 mg cholic acid (sodium salt),and 60 mg dithiothreitol. The mixtures were shaken 16 hours at 37° C.After 16 hours, the samples were centrifuged at 3,000 RPM for 20minutes. To 0.9 ml of supernatant removed after centrifugation was added10 μl of phenylmethylsulfonyl fluoride (6% in ethanol). This was mixed,followed by the addition of 90 μl CuSO₄.5H₂O (10 gm/liter). The mixtureswere shaken at 37° C. for 30 minutes.

Six grams of dry A-25-120 DEAE Sephadex® available from Sigma ChemicalCo. of St. Louis, Mo., was added to 100 ml of distilled, deionizedwater. The suspension was allowed to swell overnight in the refrigerator(2-8° C). After swelling, the suspension was placed in a beaker with alarge magnetic stirring bar, and was vigorously stirred on a magneticstirring plate for 60 minutes until the resin had broken up into smallparticles resulting in a very fine suspension.

To 400 μl of each of the hair digest solutions was added 300 μl of the6% resin suspension in a glass tube. The glass tubes were rotatedvigorously (greater than 200 rotations/minute) for 30 minutes in such away as to keep the resin suspended and in contact with the digestsolution. To each of the tubes was added 0.7 ml distilled water. Thetubes were then centrifuged, and 1.0 ml of the supernatant from theresinated digest solutions was removed. 200 μl of 1M phosphate bufferwas then added to each solution. 100 μl of the filtrate of each samplewas then assayed for marijuana by RIA. The results were as follows:

Sample 1 (known negative) 4597 cpm

Sample 2 (known positive) 2683 cpm

Sample 2 demonstrated a B/B_(o) of 59% as compared to negative samples(100%), a positive result for the presence of marijuana analyte inSample 2.

EXAMPLE 12

Known Negative Hair Samples Compared to Known Positive Hair SamplesUsing Ion Exchange Resin

Eight negative hair samples known to show great variability in bindingin the marijuana assay due to varying amounts of interfering crossreacting substance, and a hair sample from a known marijuana user weredigested and neutralized by the procedure described in Example 10. Thedigests were treated with water suspensions of various resins preparedin the concentrations (as percent of bottled resin relative to totalsuspension) shown below and the resinated digest solution was thentested in RIA.

In more detail, 0.4 ml of the digest solution was mixed 0.3 ml of theresin suspension in a 13×100-mm glass tube, and the tubes were shakenat >200 rpm for 30 minutes. Then 0.75 ml of distilled water was added toeach of the tubes, and the tubes were mixed well and centrifuged at 200RPM for 10 minutes. One ml of supernatant was removed from each andplaced in a 13×100 mm glass tube.

In performing the marijuana assay, 200 μl of 1 M, pH 7 phosphate bufferwas added to the tube, followed by 100 μl I¹²⁵ marijuana tracer and 100μl anti-cannabinoid antibody. The tubes were mixed and incubated 1 hourat room temperature. 200 μl of a second precipitating antibody was addedfollowed by 1 ml 6% polyethylene glycol. After 30 minutes at roomtemperature, the tubes were centrifuged at 3000 RPM in a refrigeratedcentrifuge. After decanting, the tubes were counted in a gammaspectrometer.

The means and standard deviations of the counts of the negatives werecalculated to determine the effectiveness of the resin in removing theinterfering crossreactor substance. The counts of the positive hairsample digest divided by the mean of negatives was calculated todetermine how well the analyte was not removed.

The following results were obtained using a 2.4% resin suspension ofDEAE-Sephadex® (A-50-120):

Sample CPM Negative #1 3961 Negative #2 4391 Negative #3 3712 Negative#4 3804 Negative #5 3574 Negative #6 4004 Negative #7 4010 Negative #84252 Positive #1 2489 Positive #2 2424

The mean of the negative samples was 3964, with a standard deviation of238 (6% of the mean). The B/B_(o) of the positive samples is 62%. Thus,the signal to noise ratio indicates that the assay is sensitive.

For DEAE Sepharose® (CL-6B), the following 5 results were obtained usinga 24% resin suspension:

Sample CPM Negative #1 4085 Negative #2 4070 Negative #3 3661 Negative#4 3771 Negative #5 3555 Negative #6 3677 Negative #7 3806 Negative #84137 Positive #1 2550 Positive #2 2539

The mean of the negative samples was 3845, with a standard deviation of196 (5% of the mean). The B/B_(o) is 66%. Thus, again, the signal tonoise ratio indicates that the assay is sensitive.

While there have been described what are presently believed to bepreferred embodiments of the invention, it will be apparent to oneskilled in the art that numerous changes can be made in the ingredients,conditions and proportions set forth in the foregoing embodimentswithout departing from the invention as described herein and as definedin the appended claims.

What is claimed is:
 1. A method for the detection and identification ofan organic analyte from the bloodstream of a subject which becomesembedded in a keratinized structure of the subject which comprises: (a)preparing a mixture comprising an enzyme suitable for digestion of thekeratinized structure, an agent selected from the group consisting ofdithiothreitol and dithioerythritol, and a sample of the keratinizedstructure; (b) permitting the digestion of the sample; and (c)subjecting a portion of the mixture to direct analysis by immunoassay todetermine whether an organic analyte is present in the sample.
 2. Themethod according to claim 1 wherein the keratinized structure is hair.3. The method according to claim 1 wherein the pH at which the method isperformed is between about 6 and
 8. 4. A method for the detection andidentification of an organic analyte from the bloodstream of a subjectwhich becomes embedded in the hair of the subject which comprises: (a)preparing a mixture comprising an enzyme suitable for digestion of thehair, an agent selected from the group consisting of dithiothreitol anddithioerythritol, and a sample of the hair; (b) permitting the digestionof the sample; and (c) subjecting a portion of the mixture to directanalysis by immunoassay to determine whether an organic analyte ispresent in the sample.